Epidermal stem cells are of great importance because they play a central role in tissue homeostasis, wound repair (both delayed in aging), as well as in tumor initiation, and for gene therapy. Studies of epidermal stem cells have been hampered by a lack of markers by which to identify them. Studies of putative markers have in turn been limited by lack of adequate functional stem cell assays. Studies of epidermal stem cells performed to date rely on relatively short-term in vitro colony forming units as a tool to assess ?stemness?. However, studies in other tissues have shown that in vitro colony forming units do not represent the true long-term repopulating cell. Borrowing from well-established functional assays in hemopoietic cells, we have designed a novel in vivo competitive repopulating assay to study stem cell function in epidermal cells. Our assay utilizes green fluorescent protein (GFP) to distinguish epidermally derived test cells from non-green competitor cells. These keratinocytes produce an epidermis when seeded in a chamber on the back of a non-GFP mouse. A limiting dilution technique allows the absolute number of stem cells in a keratinocyte population to be determined using this assay. This assay has two key features. The first is the use of a long-term competitive assay that ensures not only the detection of the most primitive epidermal stem cells but also the survival of the transplanted keratinocytes, even when seeded with very low numbers of test cells. The second is the use of a limiting dilution design to allow stem cell quantification. Our long-term objective is to elucidate the effects of aging on epidermal stem cells, and determine the mechanism(s) by which the delayed epidermal homeostasis and wound healing may be improved. The specific aims include, first, to fully optimize our assay, to enable us to assess aged and young murine epidermal stem cells. We will then compare the absolute number of stem cells in aged vs. young epidermis, and determine the proliferative potential of an equal number of stem cells from young vs. aged epidermis. This work will allow us to begin to understand the changes in epidermal stem cells with age, and may point to further studies to improve epidermal cell function and/or wound healing in the aged. These studies will also provide the first functional in vivo assay for epidermal stem cells, an important step for analvzing (T putative markers for this elusive epidermal cell, for cutaneous stem cell-targeted gene therapy, and for more basic studies of epidermal stem cell regulation and differentiation.